The overall goal of this grant application is to clarify the structure and the mechanism of action of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans. During the current N- terminal amino acid sequence of the NADH-binding subunit of this enzyme complex. Complete DNA sequencing of the gene cluster has been carried out. It is composed of 18,106 base pairs and contains 14 structural genes (designated N01-14) and 6 unidentified reading frames (designated URF1-6). Furthermore, we have expressed the NQ02 gene encoding the putative iron- sulfur 25-kDa subunit in E. coli. The expressed 25kDa subunit bears a single binuclear iron-sulfur cluster with rhombic symmetry (gx,y,z=1.92, 1.95, and 2.00). EPR, resonance Raman and magnetic CD spectroscopic studies indicate a striking similarity between the properties of the [2Fe- 2S] cluster in the 25-kDa subunit and those of the subclass of ferredoxin- type [2Fe-2S] centers typified by Clostridium pasteuriamum binuclear ferredoxin. Utilizing site-directed mutagenesis, we have verified that the conserved four cysteine residues (C96, C101, C137, and C141) coordinate the [2Fe-2S] cluster in the 25-kDa subunit. Studies planned for this grant period are as follows: (i) Characterization of the remaining putative NADH and iron-sulfur cluster binding subunits (NQ01, 3, 6, and 9) and determination of amino acid residues involved in coordination of prosthetic groups will be carried out. (ii) Overexpression and characterization of the putative non-cofactor binding, water soluble subunits (NQ04 and 5) will be carried out. The peripheral subcomplex of the Paracoccus NDH-1 will be reconstituted with the expressed subunits. (iii) The stoichiometry of the hydrophobic subunits (NQ07, 8, 10 - 14) will be determined. Their topology in situ will be investigated by immunochemical and molecular biological methods. (iv) The URF1-6 products will be chemically identified. Function roles of the URF products will be clarified by using gene deletion techniques. (v) Studies on effects of various culture conditions on the biosynthesis of the Paracoccus NDH-1 will be performed.